Dominique G. Rouiller

Activation of NF-κB by Extracellular Matrix Is Involved in Spreading and Glucose-stimulated Insulin Secretion of Pancreatic Beta Cells

Laminin-5-rich extracellular matrix derived from 804G cells (804G-ECM) engages β1 integrins to induce spreading, improve glucose-stimulated insulin secretion (GSIS), and increase survival of pancreatic beta cells. The present study examines whether 804G-ECM activates the transcriptional activity of NF-κB and the involvement of NF-κB in those effects of 804G-ECM on pancreatic beta cells. 804G-ECM induces nuclear translocation and the DNA binding activity of the p65 subunit of NF-κB. 804G-ECM-induced nuclear translocation of NF-κB was weak as compared with that induced by interleukin-1β. Transient 804G-ECM-induced DNA binding activity of NF-κB (peak at 2 h) and overexpression of NF-κB target genes IκBα and NF-κB1(p105) (peak at 4 h) were observed. When NF-κB was inhibited by an inhibitor of IκBα phosphorylation (Bay 11-7082) or by a recombinant adenovirus expressing the nonphosphorylatable form of IκBα, 804G-ECM-induced cell spreading and actin cytoskeleton organization were reduced. GSIS from cells on 804G-ECM was inhibited 5-fold, whereas cell survival was not affected. In summary, the results indicate that 804G-ECM induces a transient and moderate NF-κB activity. This study shows for the first time that ECM-induced NF-κB activity is necessary in maintaining GSIS, although it does not affect survival of pancreatic beta cells. The effects of ECM-induced NF-κB activity contrast with the deleterious effects of cytokine-induced NF-κB activity. It is proposed that transient and moderate NF-κB activity is essential for proper function of the pancreatic beta cell.

Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet cell types

Cells within rat islets of Langerhans are typically organized as a core of B-cells, surrounded by the other cell types. When mixed in culture, primary islet cells and insulinoma (RIN2A) cells form aggregates where B-cells are centrally located, surrounded by non-B-cells, while RIN-cells segregate as the outermost layer. To gain insight into the molecular basis underlying this nonrandom cellular organization, the aggregation properties of the three cell populations were studied. Isolated islet cells were separated into B-cells and non-B-cells by autofluorescence-activated cell sorting (FACS). In a short-term aggregation assay, primary B-cell aggregation in the absence of calcium was only 19 +/- 3.7%, compared to the 67 +/- 2.9% seen in the presence of calcium (mean +/- SEM; P less than 0.001; n = 7). By contrast, non-B-cell aggregation and RIN cell aggregation in the absence of calcium (62 +/- 2 and 66 +/- 2%, respectively) were only slightly less than with calcium (70 +/- 3 and 76 +/- 3%). The surface density of the Ca2(+)-independent neural CAM (NCAM) was therefore measured by flow cytometry and found to be 2.64 +/- 0.82-fold higher in non-B-cells, compared to that in B-cells (P less than 0.01; n = 3). Even higher levels were found on RIN cells. In the three cell types, NCAM-140 was the only molecular form detected by immunoblotting. In conclusion, differences in the calcium dependency of aggregation and in the levels of NCAM are demonstrated among islet B-cells, non-B-cells, and RIN cells. Because cell-cell adhesion is crucial for the maintenance of adult tissue, these aggregation specificities might contribute to the concentric segregation of islet cell types in culture and to the nonrandom distribution of cells within rat islets.

Blockade of β1 Integrin–Laminin-5 Interaction Affects Spreading and Insulin Secretion of Rat β-Cells Attached on Extracellular Matrix

When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic β-cells spread in response to glucose and secrete more insulin compared with cells attached on poly-l-lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor β1 integrin are implicated in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G matrix responsible for the effect of 804G matrix on β-cell function and spreading. When expression of two well-known laminin-5 ligands, β1 and β4 integrin, was assessed by Western blot and RT-PCR, only the β1 integrin was detected in β-cells. Anti–β1 integrin antibody reduced the spreading of β-cells on 804G matrix. Blockade of the interaction between β1 integrins and laminin-5 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti–β1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti–β1 integrin and –laminin-5 antibodies interfere with spreading of β-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling via engagement of β1 integrins by laminin-5 is an important component of normal β-cell function.

Tumor necrosis factor-alpha modifies adhesion properties of rat islet B cells.

The characteristic three-dimensional cell type organization of islets of Langerhans is perturbed in animal models of diabetes, suggesting that it may be important for islet function. Rat islet cells in culture are able to form aggregates with an architecture similar to native islets (pseudoislets), thus providing a good model to study the molecular basis of islet architecture and its role in islet function. Sorted islet B cells and non-B cells were permanently labeled with two different fluorescent dyes (DiO and DiI), mixed, and allowed to form aggregates during a 5-d culture in the presence or absence of TNF-alpha (100 U/ml), a cytokine suggested to be implicated in the early physiological events leading to insulin-dependent diabetes mellitus. Confocal microscopy of aggregates revealed that TNF-alpha reversibly perturbs the typical segregation between B and non-B cells. Insulin secretion, was altered in the disorganized aggregates, and returned towards normal when pseudoislets had regained their typical architecture. The homotypic adhesion properties of sorted B and non-B cells cultured for 20 h in the presence or absence of TNF-alpha were studied in a short term aggregation assay. TNF-alpha induced a significant rise in Ca(2+)-independent adhesion of B cells (from 24 +/- 1.1% to 44.3 +/- 1.2%; n = 4, P < 0.001). These findings raise the possibility that the increased expression of Ca(2+)-independent adhesion molecules on B cells leads to altered islet architecture, which might be a factor in the perturbation of islet function induced by TNF-alpha.

Insulin inhibits somatostatin-like immunoreactivity release stimulated by intragastric HCL

To determine the effect of an increase in insulin levels within the range occuring under physiologic conditions on the protein- and acid-induced release of splanchnic somatostatin, insulin was infused in dogs for 1 h following the intragastric instillation of a neutral protein load (20% liver extract at pH 7), a weak stimulus of somatostatin-like immunoreactivity (SLI), and after an intragastric HCI, a strong stimulus of SLI release, instilled 30 min later. Insulin levels between 50 and 60 μU/ml significantly reduced the rise in peripheral venous SLI levels elicited by the acid load from a mean integrated incremental value of 1705 ±182 pg/ml in controls to 840 ± 312 in the insulininfused group (P < 0.05). Prevention of the insulininduced hypoglycemia and the secondary rise in glucagon, a known stimulus of pancreatic somatostatin secretion, by means of a concomitant infusion of glucose, did not modify the reduction in acid-induced increase in plasma SLI concentration associated with hyperinsulinemia. Insulin-glucose infusion significantly lowered the SLI in the pancreaticoduodenal vein, and in the gastroepiploic vein draining the antrum (P < 0.02; P < 0.05), but not in the short gastric veins draining the fundus of the stomach in response to the acid load. It is concluded that a physiologic elevation of insulin levels causes significantly reduced response of SLI to an intragastric acid load in dogs. This reduction is explained by a diminished increment of SLI in the venous effluent of the pancreas and the antrum.

Epidermal growth factor receptor internalization and biosynthesis in the diabetic rat

The number of surface EGF receptors as well as their internalization rate and biosynthesis were analyzed in hepatocytes freshly isolated from control, streptozotocin-diabetic, and insulin-treated diabetic rats. All three parameters were decreased in diabetic animals and values were corrected by insulin treatment. Moreover, the inhibition of synthesis was specific for the EGF receptor since the other biosynthetically labeled proteins were not affected. These data demonstrate that the reduced number of hepatocyte surface EGF receptors results from an inhibition of EGF-receptor synthesis which is not compensated by a reduced internalization rate.